The studies described in this proposal are directed at determining the biochemical mechanism of genetic recombination. The focus of this proposal is on the Escherichia coli recA protein and on the contributions that other recombinationally important proteins such as SSB protein, recBCD enzyme, recQ helicase, and recJ nuclease as well as other helicases, topoisomerases, and polymerase make to the pairing of homologous DNA sequences and to the exchange of DNA strands. These proteins catalyze the biochemical steps that are of fundamental importance to the process of homologous recombination, and an understanding of how these steps occur is essential for a complete understanding of the events that comprise the cellular process. This grant proposal has two broad research objectives. The first is to further understand the biochemical mechanism by which recA protein facilitates both the recognition of DNA sequence homology and the subsequent exchange of DNA strands between two homologues. Since the recA protein plays such a central role in recombination and because it serves as the prototype for all protein- promoted DNA strand exchange processes, further study is essential. The second objective is to both reconstitute and analyze in vitro as system, assembled from purified components, capable of carrying out homologous recombination. By taking advantage of in vitro DNA pairing and DNA strand exchange reactions between defined DNA substrates and of purified proteins now available, it is possible to reproduce some of the individual steps that comprise homologous recombination. Using these assays, the ability of recA protein to bind preferentially to DNA structures that might be intermediates in the homologous pairing process or to specific DNA sequences will be examined. The mechanism and specificity determinants of homology search process will also be determined. The role of some helicases is presumably to serve as initiators of the recombination process by unwinding duplex DNA. This has been established for recBCD enzyme and will be tested for recQ protein. Yet other proteins might act to stabilize nascent paranemic joint molecules that are formed by recA protein: this could include topoisomerases, nucleases, and DNA polymerases. Finally, the role of these proteins in resolution of crossover structures will be examined. Knowledge of the mechanism of aberrant chromosomal translocations and may also provide new experimental approaches for gene targeting therapies.
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